Zenon blocking reagent

To access the percentage of cells infected by ZIKV, cells were detached with 2.5 % trypsin-EDTA and fixation and permeabilization were performed using commercial flow cytometry kits (e-Bioscience, San Diego, CA, EUA). A monoclonal primary mouse antibody anti-ZIKV NS1 (1:100, E107, MA5-24585, Invitrogen) was incubated with Zenon Alexa 488 conjugated secondary anti-mouse antibody (1:250; Thermo Fisher Scientific) for 5 min to form an immunocomplex with further addition of Zenon Blocking Reagent for further 5 min. The immunocomplex was later incubated with cells for 1 h at 37 °C. The percentage of ZIKV infected cells was analysed with FACS CantoTM® II flow cytometer (BD Biosciences, San Jose, CA, USA), using the software FlowJo (TreeStar, Ashland, OR, USA).

All culture groups were fixed with 4 % paraformaldehyde in PBS and permeabilized with 0.1 % Triton X-100 in PBS (v/v). Phalloidin-fluorescein (FITC) conjugated staining (1:100; Abcam, Cambridge, UK). Then, it was performed unspecific antigens blockade with 0.05 % fish skin gelatin (Merck/Sigma-Aldrich, Germany) in PBS for 1 h. It was followed by monoclonal primary mouse antibody anti-ZIKV NS1 (1:100, E107, MA5−24585, Invitrogen, UK) incubation with Zenon Alexa 594 conjugated secondary anti-mouse antibody (1:250; Thermo Fisher Scientific) for 5 min to form an immunocomplex, which was later incubated with Zenon Blocking Reagent (1:250; Thermo Fisher Scientific, Wathlam, MA, USA) for further 5 min at room temperature. The immunocomplex mixture was added to cells for 1 h at 37 °C. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:1000 in PBS; Merck/Sigma-Aldrich), and mounting was performed with PBS/glycerol (1:9, v/v) under glass slides. The results were visualized with a fluorescence microscope Nikon DS-Ri1 (Nikon, Japan) and images were acquired using the DP2-BSW software (Nikon).